The present invention is directed to compositions containing one or more agents that recognize the active conformation of human von Willebrand and that inhibit the interaction between this factor and platelets. The compositions can be used either therapeutically or prophylactically to prevent thrombus formation in patients. Compositions may also be used diagnostically to detect sites where thrombosis is likely to occur.
Thrombosis occurring at atherosclerotic plaques is a major cause of morbidity and mortality in the United States. The initial event in thrombus formation is the adhesion of platelets to an injured or diseased arterial wall. Adhesion is enhanced and stabilized by a plasma protein, the von Willebrand Factor, which forms a bridge between components of the vessel wall and receptors on the platelet surface, primarily the glycoprotein Ib/IX/V complex. There are two unique features of this interaction that set it apart from adhesive events involving other cells and plasma proteins. First, the interaction of vWF with platelets is the only reaction that permits platelets to remain attached to vessel walls under the high shear/high flow conditions present in arteries, arterioles, and capillaries. Second, vWF is the only plasma adhesive protein which must undergo a conformation change before it is able to bind to its platelet receptor.
Attempts have been made to prevent thrombus formation by introducing either antibodies (see, e.g., EP 747,060; WO 96/17078; and U.S. Pat. No. 5,336,618) or peptides (see, e.g., U.S. Pat. No. 5,688,912; U.S. Pat. No. 5,493,007; U.S. Pat. No. 5,114,842; WO 93/16712; and EP 319,315) thatbind to platelet receptors. One problem with the use of such agents is that they are nonselectivexe2x80x94interfering with the function of all circulating platelets. The development of an agent that acts specifically at sites of thrombosis to inhibit platelet adhesion would represent a clear advance in the treatment and prevention of stroke, myocardial infarction, and related conditions. Equally important, such an agent might be used diagnostically to identify sites where blood vessels are at risk of becoming occluded.
Using recombinant DNA and phage display technology, murine anti-human vWF antibodies have been made which specifically recognize activated vWF and interfere with its ability to promote platelet adhesion. The antibodies act at sites of thrombus formation but do not bind to circulating, unactivated forms of vWF. This results in antithrombotic agents that are both safer and more efficacious.
In its first aspect, the invention is directed to a composition comprising an antibody that binds selectively to the active conformation of human vWF, thereby inhibiting its ability to interact with platelets. As used herein, xe2x80x9cselective bindingxe2x80x9d means that an antibody has at least a tenfold, and preferably at least a hundredfold, greater affinity for vWF when it is in its active conformation compared to when it is unactivated. Relative affinity can be determined using standard binding assays in which vWF is examined both in the presence and absence of an activating agent such as Ristocetin. Unless otherwise indicated, the term xe2x80x9cantibodyxe2x80x9d refers both to intact antibodies as well as to fragments, particularly to recombinantly engineered fragments, that retain their ability to bind to antigen. Inhibition of platelet binding occurs whenever there is a statistically significant reduction in the amount of vWF-induced platelet aggregation in the presence of antibody. In the most preferred embodiment, compositions contain recombinantly produced single chain variable region (ScFv) fragments of immunoglobulins directed against a vWF-A1 epitope. Typically, the ScFv fragment will be derived from the mouse and compositions designed for therapeutic administration will contain a pharmaceutically acceptable carrier.
In a second aspect, the invention is directed to a method of identifying an ScFv fragment that binds selectively to the active conformation of human vWF. The method involves immunizing an animal, preferably a mouse, with an immunogen (either a peptide or a nucleic acid encoding a peptide) derived from the A1 region of human vWF. After immunization, mRNA is isolated from the animal and used to produce an ScFv cDNA library in a bacteriophage capable of displaying the fragments. The library is then screened to identify phage expressing a fragment that binds selectively to the active conformation of vWF. Binding may be determined directly, in the presence and absence of an agent inducing vWF to assume an active conformation, or by examining the inhibition of vWF-induced platelet aggregation. Once an appropriate phage has been identified, the DNA encoding the ScFv fragment may be recovered and subcloned in an expression vector. Finally, recombinant ScFv is produced in a host cell transformed with the vector and purified. The ScFv fragments obtained in this manner are part of the invention.
The present invention is also directed to a method for preventing thrombus formation in a patient by administering a pharmaceutical composition containing an antibody of the type discussed above, i.e., an antibody binding selectively to the active conformation of human vWF. The pharmaceutical composition should be administered at a dosage sufficient to prevent the binding of activated vWF to platelets and may be administered either therapeutically or prophylactically. Therapeutically, the composition may be administered to a patient with an occluded blood vessel either alone or in conjunction with thrombolytic agents such as tissue plasminogen activator or streptokinase. Prophylactically, the compositon may be administered to patients at risk of thrombosis due to atherosclerosis or during medical procedures that carry a risk of vessel occlusion, e.g., angioplasty, stent placement, or graft insertion.
Antibodies may also be detectably labeled and used in conjunction with imaging techniques to determine sites within the vasculature where thrombosis is likely to occur, e.g., where there has been plaque rupture or blood vessel damage. Because ScFv fragments are missing regions of antibodies that are often responsible for nonspecific binding, these fragments are preferred for all in vivo diagnostic procedures.